Method for the treatment or prevention of osteoarthritis

ABSTRACT

The present invention is in the field of medicine and provides means and methods for the treatment, prevention or amelioration of osteoarthritis. More in particular, it provides a peptide for use in the treatment, amelioration or prevention of osteoarthritis, wherein the peptide is between 12 and 28 amino acids in length and comprises an amino acid sequence according to SEQ ID NO: 16 or a variant thereof according to formula 2, wherein the amino acid sequence of said peptide is comprised in SEQ ID NO: 34 or a variant thereof according to formula 1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/092,671, filed Oct. 10, 2018, which is a national phase entry under35 U.S.C. § 371 of International Patent Application PCT/EP2017/057718,filed Mar. 31, 2017, designating the United States of America andpublished in English as International Patent Publication WO 2017/178251on Oct. 19, 2017, which claims the benefit under Article 8 of the PatentCooperation Treaty to European Patent Application Serial No. 16165093.2,filed Apr. 13, 2016, the disclosure of each of which is herebyincorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

The present invention is in the field of medicine and provides means andmethods for the treatment, prevention or amelioration of osteoarthritis.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA) is a type of joint disease that results frombreakdown of joint cartilage and underlying bone, combined with anoverall joint pathology including synovial inflammation and—fibrosis andmeniscus pathology. The most common symptoms are joint pain andstiffness. Initially, symptoms may occur only following exercise, butover time may become constant. Other symptoms may include jointswelling, decreased range of motion, and when the back is affected by OAweakness or numbness of the arms and legs may present. The most commonlyinvolved joints are those near the ends of the fingers, at the base ofthe thumb, neck, lower back, knees, and hips. Joints on one side of thebody are often more affected than those on the other. Usually theproblems come on over years. It can affect work and normal dailyactivities. Unlike other types of arthritis, only the joints aretypically affected.

Causes include previous joint injury, abnormal joint or limb developmentabnormal alignment of joints and inherited factors. Risk is greater inthose who are overweight, have one leg of a different length, and havejobs that result in high levels of joint stress. Osteoarthritis isbelieved to be caused by mechanical stress on the joint and low gradeinflammatory processes. It develops as cartilage is lost with eventuallythe underlying bone becoming affected. The subchondral bone is alsothought to be crucially involved in the etiology of the disease. As painmay make it difficult to exercise, muscle loss may occur. Diagnosis istypically based on signs and symptoms with medical imaging and othertests occasionally used to either support or rule out other problems.Unlike in rheumatoid arthritis, which is primarily an inflammatorycondition, the joints do not typically become hot or red.

Treatment includes exercise, efforts to decrease joint stress, supportgroups, joint lubrication and (local) pain medications. Efforts todecrease joint stress include resting and the use of a cane. Weight lossmay help in those who are overweight. Pain medications may includeparacetamol (acetaminophen). If this does not work, orally administeredNSAIDs such as naproxen may be used, or locally administeredcorticosteroids (eg triamcinolonacitonide) may be used, but thesemedications are associated with greater side effects. Opioids if usedare generally only recommended short term due to the risk of addiction.If pain or movement restriction interferes with normal life despiteother treatments, joint replacement surgery may help. An artificialjoint, however, only lasts a limited amount of time and total jointreplacement surgery is associated with severe complications likeosteomyelitis. Outcomes for most people with osteoarthritis are goodafter total joint replacement.

OA is the most common form of arthritis with disease of the knee and hipaffecting about 3.8% of people as of 2010. Among those over 60 years oldabout 10% of males and 18% of females are affected. It is the cause ofabout 2% of years lived with disability. In Australia about 1.9 millionpeople are affected, and in the United States about 27 million peopleare affected. Before 45 years of age it is more common in men, whileafter 45 years of age it is more common in women. It becomes more commonin both sexes as people become older.

While OA is a degenerative joint disease that may cause gross cartilageloss and morphological damage to other joint tissues, more subtlebiochemical changes occur in the earliest stages of OA development. Thewater content of healthy cartilage is finely balanced by compressiveforce driving water out & swelling pressure drawing water in, supportedby a distinct osmotic tissue pressure. Collagen fibers exert thecompressive force, whereas the Gibbs-Donnan effect & cartilageproteoglycans create osmotic pressure which tends to draw water in.

However, during onset of OA, the collagen matrix becomes moredisorganized and there is a decrease in proteoglycan content withincartilage. The breakdown of collagen fibers results in a net increase inwater content. This increase occurs because whilst there is an overallloss of proteoglycans (and thus a decreased osmotic pull) it isoutweighed by a loss of collagen. Without the protective effects of theproteoglycans, the collagen fibers of the cartilage can becomesusceptible to degradation and thus exacerbate the degeneration.Inflammation of the synovium (joint cavity lining) and the surroundingjoint capsule can also occur, though often mild (compared to what occursin rheumatoid arthritis).

Changes in the articular cartilage and articular chondrocytes thatcharacterize OA resemble the cellular developmental process drivingskeletal development by endochondral ossification. The analogy betweenendochondral ossification and OA progression has been widely recognized.Many of the cartilage-degrading enzymes that are excreted byhypertrophic chondrocytes in the growth plate are also central inprogression and worsening of the OA condition. Also, well-known pathwayscontrolling chondrocyte differentiation in the growth plate (RUNX2,COL10A1, ALP) are found active or deregulated in OA articularchondrocytes as well.

Other structures within the joint can also be affected. The ligamentswithin the joint become thickened and fibrotic and the menisci canbecome damaged and wear away. Menisci can be completely absent by thetime a person undergoes a joint replacement. New bone outgrowths, called“spurs” or osteophytes, can form on the margins of the joints, possiblyin an attempt to improve the congruence of the articular cartilagesurfaces in the absence of the menisci. The subchondral bone volumeincreases and becomes less mineralized (osteoporotic/osteopenic). Allthese changes can cause problems in functioning and mechanical supportfor the overlying cartilage layer. The pain in an osteoarthritic jointhas been related to thickened synovium and subchondral bone lesions.

Biochemically, OA is characterized by synthesis of extracellular matrix(ECM)-degrading enzymes, such as aggrecanases (a disintegrin andmetalloproteinase with trombospondine motifs (ADAMTSs)) and matrixmetalloproteinases (MMPs), resulting in the active breakdown of thecartilage tissue matrix. The analogy between endochondral ossificationand OA progression has been recognized and many of the cartilagedegrading and mineralization enzymes that are secreted by hypertrophicchondrocytes in the growth plate are also crucially involved in OA.

Notwithstanding the progress made in the understanding of diseasemechanisms, established and experimental treatment of OA is mainlysymptomatic by alleviating pain and interfering with the cartilagedegenerative processes to postpone total joint replacement.

SUMMARY OF THE INVENTION

The invention relates to a peptide for use in the treatment,amelioration or prevention of osteoarthritis, wherein the peptide isbetween 12 and 28 amino acids in length and comprises an amino acidsequence according to SEQ ID NO: 16 or a variant thereof according toformula 2, wherein the amino acid sequence of said peptide is comprisedin SEQ ID NO: 34 or a variant thereof according to formula 1.

Formula 1: (SEQ ID NO: 47)X1 X2 X3 G Y X4 A X5 Y S E G X6 S X7 X8 X9 L X10 X11 X12 M N A T X13 H A

wherein

X1=A or I or L or M or Y or V or E or H or K or Q or R,

X2=P or Y or M,

X3=E or R or H or K or N or P or Q or S or T or I or L or M or V,

X4=A or E or Q or R or S,

X5=Y or N or D,

X6=E or A or Q,

X7=A or D or E or H or K or S,

X8=F or A or D or E or H or Q or R or S,

X9=P or M,

X10=N or A or D or S or T or E or Q or R or I or V,

X11=S or A or D or E or H or K or N or P or Q or T,

X12=Y or H or D or G or H or N or R or S or T or wherein

X13=N or F or W or Y or H or K or R.

Formula 2: (SEQ ID NO: 48) Y S E G X6 S X7 X8 X9 L X10 X11

wherein

X6=E or A or Q,

X7=A or D or E or H or K or S,

X8=F or A or D or E or H or Q or R or S,

X9=P or M,

X10=N or A or D or S or T or E or Q or R or I or V or wherein

X11=S or A or D or E or H or K or N or P or Q or T.

DETAILED DESCRIPTION OF THE INVENTION

Osteoarthritic chondrocytes display a typical phenotype that ischaracterized by decreased expression of chondrogenic genes SOX9,COL2A1, ACAN and BAPX1/NKX3.2 and an increased expression ofhypertrophic genes RUNX2, COL10A1, ALP, genes encoding cartilage matrixdegrading enzymes MMP13, ADAMTS5 and inflammatory genes COX-2 and IL-6(FIG. 1). We show herein that a protein called bone morphogeneticprotein-7 (BMP-7, also called OP-1) is capable of rescuing the OAphenotype (FIG. 1). Ours and earlier studies addressing thedisease-modifying properties of BMP-7 show that it decreases MMP13expression in IL-1β-exposed chondrocytes, stimulates proteoglycansynthesis in OA chondrocytes, counteracts inflammatory cytokines (e.g.IL-1β) and induces an anabolic response in healthy chondrocytes.Intra-articular administration of BMP-7 protects against OA developmentand delays progression of OA in rats. A phase-1 clinical trial has beencompleted in OA patients and reported no serious adverse events afterintra-articular injection of BMP-7. In congruency with this, the datareported herein unveil that BMP-7 actively suppresses the (OA)chondrocyte hypertrophic phenotype (FIG. 1).

Despite these promising results, intra-articular use of full-lengthrecombinant human BMP-7 for OA-treatment may be at risk for clinicaluse. Pre-clinical testing showed that weekly intra-articular BMP-7injections were necessary to acquire a relevant result. This highfrequency of intra-articular injections is not acceptable for clinicaluse due to risk of septic arthritis and patient discomfort. While asolution to prevent frequent intra-articular injections would encompassthe encapsulation of BMP-7 in an intra-articular release system forlong-term controlled release, retaining bioactivity of BMP-7 will posean enormous challenge due to the denaturing conditions that generallyapply for the production process of currently existingcontrolled-release systems. OA synovial fluid, in which BMP-7 is likelyto be delivered, is a harsh hydrolytic and proteolytic environment thatis expected to cause rapid degradation of the administered BMP-7.Finally, production of GMP-grade BMP-7 is technologically demanding withaccompanying high costs.

To safeguard and enable the clinical use of the highly favourablecharacteristics of BMP-7 activity for OA-treatment, we sought for BMP-7molecular mimics that are better compatible with the harsh OA synovialfluid environment and can potentially be incorporated in intra-articularmolecular release systems for long-term release. For that reason we setout to prepare a set of overlapping 20-mer peptides (table 1) thatcollectively cover the entire mature BMP-7 polypeptide of 139 aminoacids (table 4). The results confirmed earlier findings [20,21] in thatnone of the peptides mimicked the potential of BMP-7 to rescue the OAphenotype. In more detail: all peptides shown in table 1 were tested forexpression of genes ALP, MMP13, ADAMTS5, COL10A, COX-2, BAPX1 NKX3.2 andRUNX2 and found to evoke pro-hypertrophic, pro-mineralizing,pro-katabolic and pro-inflammatory responses. This was not unexpected,since pro-mineralizing/pro-osteogenic peptides have been described fromBMP-2 [20] and BMP-7 [21, 31].

TABLE 4 Amino acid sequence of mature BMP-7, Cysteineand Serine residues underlined. Name Amino Acid Sequence SEQ ID NO:Mature BMP-7 STGSKQRSQN RSKTPKNQEA 45 LRMANVAENS SSDQRQA C KKHELYVSFRDL GWQDWIIAPE GYAAYY C EGE  C AFPLNSYMN ATNHAIVQTL VHFINPETVP KPCC APTQLN AISVLYFDDS SNVILKKYRN MVVRA C G C H Mature BMP-7STGSKQRSQN RSKTPKNQEA 46 with Cysteine LRMANVAENS SSDQRQA S KKreplaced by HELYVSFRDL GWQDWIIAPE Serine GYAAYY S EGE  S AFPLNSYMNATNHAIVQTL VHFINPETVP KP SS APTQLN AISVLYFDDS SNVILKKYRN MVVRA S G S H

Surprisingly, however, when we replaced the cysteine residues in BMP-7with serine residues, our data unveiled the presence of a highly definedregion within BMP-7 that indeed harbors peptides with the sought-afterBMP-7-mimicking activity.

Peptides from this region are designated as “region-A peptides” hereinfurther. Consecutive peptides from this region caused significantlydecreased expression of OA chondrocyte phenotypic markers COL10A1, ALP,RUNX2, ADAMTS5, IL-6, MMP13 and COX-2, as well as increased expressionof chondrogenic markers SOX9, ACAN, BAPX1/NKX3.2 and COL2A1 in OAarticular chondrocytes.

In one aspect, the invention therefore relates to a peptide for use inthe treatment, amelioration or prevention of osteoarthritis, wherein thepeptide comprises an amino acid sequence of at least the 12 consecutiveamino acids according to SEQ ID NO: 16. This is deduced from the resultsshown in table 2.

Table 2 shows the results of expression analysis of several genes in thepresence of several peptides for use according to the invention. Furtherdetails are provided in the Examples section.

Our experiments also revealed that the peptide has a maximum length of28 amino acids or thereabout. This is deduced from the experiments shownin tables 2 and 3 wherein it is shown that the peptide is preferablyfully contained in the sequence APEGYAAYYSEGESAFPLNSYMNATNHA (SEQ ID NO:34). Every peptide that extended beyond this amino acid sequence, eitherat the NH2 terminal or the COOH terminal end, lost its activity in theassays as employed herein. Table 3 also shows some examples of peptidesfor use according to the invention.

Of course, this description should not be interpreted so narrow thatthere is no variation allowed in the peptide. The skilled person isaware of the fact that the conformation of the peptide is conserved evenwhen one or more, such as two, three, four or even five amino acids arechanged, in particular when these changes relate to conservative aminoacid substitutions. These peptides are known in the art as homologouspeptides. Hence the term “homologues” as used herein refers to peptidesthat retain their activity but differ with respect to their amino acidsequence. Homologues can be 75% identical with the sequences accordingto SEQ ID NO: 16 or SEQ ID NO: 34, or more, such as 77, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%identical.

As used herein, the degree of identity between two or more amino acidsequences is equivalent to a function of the number of identicalpositions shared by the sequences (i.e., % identity=number of identicalpositions divided by the total number of positions×100), excluding gaps,which need to be introduced for optimal alignment of the two sequences,and overhangs. The comparison of sequences and determination of percentidentity between two or more sequences can be accomplished usingstandard methods known in the art. For example, a freewareconventionally used for this purpose is “Align” tool at NCBI recoursehttp://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq

In a preferred embodiment, the alignment of two sequences is to beperformed over the full length of the polypeptides.

As used herein, the term “may” encompasses the word “can,” and the term“may be” encompasses the words “is” or “are,” depending on context.Furthermore, presence of the word “may” is intended to explain optionsfor practicing or implementing the disclosure, without limitation.

We concluded that human BMP-7-derived peptides as described herein areable to mimic the favourable characteristics of the full-length humanBMP-7 protein on the OA chondrocyte phenotype.

We have therewith determined the location and nature of the OAphenotype-suppressive action of BMP-7, while peptides from other regionsin BMP-7 displayed no activity or pro-mineralizing/-hypertrophicactions. The bioactive potency of the candidate peptides wasunexpectedly high. Independent of the tested concentration (1000, 100,10 and 1 nanoMolar (nM) were tested), almost all region-A peptidesinduced similar fold-change gene expression magnitude differences in thescreening, whereas a control peptide (SEQ ID NO: 49) did not.

BMP-7 has unique OA chondrocyte-phenotype suppressive actions (FIG. 1).This action is most pronounced when a BMP-7 concentration is used around1 nM. BMP-7 concentrations higher than 1 nM (eg 10 nM or 100 nM) haveopposite and negative effects on the chondrocyte phenotype and thusunfavorably cause increased levels of hypertrophy, increasedmineralisation, increased expression of cartilage breakdown genes andincreased expression of inflammatory genes. Without wanting to be boundby theory, we speculate that BMP-7 incorporates a dual activity that isdependent on the concentration of BMP-7.

In contrast with the BMP-7 polypeptide, the 20-mer peptides according toSEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 efficientlysuppressed the OA chondrocyte phenotype, independent of the testedconcentration (1000, 100, 10 and 1 nM were tested). With this we haveunexpectedly identified the region comprising the favorable OAphenotype-suppressive bioactivity of BMP-7, while peptides from otherregions in BMP-7 displayed pro-mineralizing, pro-hypertrophic,pro-katabolic and pro-inflammatory actions.

Next to the 24 hours to which OA chondrocytes were exposed to individualpeptides, the OA phenotype-suppressive action of the candidate peptideswas highly effective when peptides were supplemented to OA chondrocytecultures every second until day 10 in culture. Surprisingly, the OAphenotype-suppressive action of the peptides as disclosed herein evenlasted up to 8 days in culture after a single initial 48-hours exposure.

The highly potent bioactivity at nanomolar-range concentration issimilar to full-length mature BMP-7 and is a therapeutically importantdeterminant, showing that the peptides for use according to theinvention are indeed powerful BMP-7 mimics for OA therapeutical use.

In addition to the action of the peptides on OA chondrocytes, thecandidate peptides displayed biologically similar actions in an in vitromodel for chondrogenic differentiation. Remarkably, eight days of singleexposure, and even more pronounced with continuous peptide exposure ofdifferentiating chondroprogenitor cells (ATDC5), solidly decreased theexpression of hypertrophy and mineralization markers, while aggrecanexpression (healthy chondrocyte marker) and BAPX1/NKX3.2 expression(anti-hypertrophy factor) was even further increased under continuousexposure to the peptides for use according to the invention.

Taken together, the peptides for use according to the invention haveunique suppressive actions on the hypertrophic/mineralizing OAchondrocyte phenotype, suppress the expression of cartilage ECMdegrading enzymes and lower the inflammatory OA chondrocyte phenotype,while at the same time supporting a pro-chondrogenic action on OAchondrocytes and healthy chondrocytes. In contrast to the full-lengthmature BMP-7 protein, peptides may advantageously be used in therapybecause they are in general less susceptible for conformational andenzymatic inactivation. Peptides can be biochemically fine-tuned toincrease their stability and activity; functionalized for carriers; aremuch smaller and thus suitable for incorporation inapplication-dedicated release systems for long-term intra-articularrelease.

Variants of the peptides as described above may be synthesized that aremore resistant to degradation in proteolytic degradation in synovialfluid. Also, variants may be prepared that are more conformationallyrestricted and thus be more bioactive as compared to their original leadsequences. Such is well within reach for a skilled person, and requiresonly routine techniques now that the region A has been identified as theactive region for the anabolic activity of BMP-7.

Such variants may include linear peptides, linear retro inversopeptides, retro-inverso peptides, cyclic peptides, mono-looped peptides,and two-looped peptides. CLIPS technology enables the routine productionof such peptides (http://www.pepscan.com/therapeutics/clips-platform).Two-looped peptides may contain two identical peptides as describedherein or two different peptides as described herein.

All alpha amino acids but glycine can exist in either of twoenantiomers, or chiral variants, called L or D amino acids, which aremirror images of each other. While L-amino acids represent all of theamino acids found in proteins during translation in the ribosome,D-amino acids are very rarely found in eukaryotically translatedproteins. Since ribosomes are specific to L-amino acids, D-peptidesrarely occur naturally in organisms and are thus not easily digested ordegraded.

An L-peptide has three analogue sequences built from L and D aminoacids: the D-enantiomer or inverso-peptide with the same sequence, butcomposed of D-amino acids and a mirror conformation; the retro-peptide,consisting of the same sequence of L amino acids but in reverse order;and the retro-inverso or D-retro-enantiomer peptide, consisting ofD-amino acids in the reversed sequence. While the L-peptide and itsD-enantiomer are mirror structures of each other, the L-retro-peptide isthe mirror image of the D-retro-inverso-peptide. On the other hand, theL-peptide and the D-retro-inverso-peptide share a similar arrangement ofside-chains, although their carboxyl and amino groups point in opposingdirections. For small peptides that do not depend on a secondarystructure for binding, an L-peptide and its D-retro-inverso-peptide islikely to have a similar binding affinity with a target L-protein.

Peptides may be more active when they are looped, such as cyclicpeptides. Such peptides may also be more stable and resistant toproteolytic degradation.

Such peptides and polypeptides may be more stable in an environmenthostile to polypeptides, such as a hydrolytic or proteolyticenvironment. In particular for therapeutic purposes, it may beadvantageous to use more stable peptides, such as the ones mentionedabove. Hence, the invention also relates to the use of peptides selectedfrom the group consisting of linear peptides, linear retro-inversopeptides, retro-inverso peptides, cyclic peptides, mono-looped peptides,and two-looped peptides.

Ways to render a peptide less susceptible to degradation are known inthe art, such as the inclusion of one or more non-natural amino acids,such as the D-enantiomer of an L-amino acid and the retro orientation ofthe peptide backbone in the retro-inverso variant. These peptides arenon-natural, hence natural proteases are not able to cleave them.

Means and methods for increasing the bioactivity of the peptides for useaccording to the invention are also known in the art. Examples of suchtechniques are cyclization and looping of the peptides, which provides amore constraint conformational context of essential amino acid residues.

Patent application US2006/0058231 discloses BMP-7 variants with improvedproperties. This document is hereby incorporated by reference.US2006/0058231 discloses that some amino acids of BMP-7 may besubstituted with other amino acids without affecting the function of theBMP-7 protein.

Among the improved properties of BMP-7 as a result of the abovementioned amino acid substitutions are: increased expression yield,expression in the absence of a pro-domain, increased solubility,increased stability, increased specific biological activity, alteredreceptor specificity, altered receptor binding affinity, alteredco-receptor specificity, altered co-receptor binding affinity, increasedbinding to noggin, reduced binding to noggin and decreasedimmunogenicity,

With respect to the region A amino acids, this means that amino acids atpositions 1-3, 6, 8, 13, 15-17, 19-21 and 26 of SEQ ID NO: 34 may bealtered or substituted, without affecting the activity of BMP-7. In someinstances, these amendments (amino acid substitutions), even improvedthe activity of BMP-7.

The region corresponding to SEQ ID NO: 34 may therefore also bedescribed as shown in formula 1:

Formula 1: (SEQ ID NO: 47)X1 X2 X3 G Y X4 A X5 Y S E G X6 S X7 X8 X9 L X10 X11 X12 M N A T X13 H A

wherein

X1=A or I or L or M or Y or V or E or H or K or Q or R,

X2=P or Y or M,

X3=E or R or H or K or N or P or Q or S or T or I or L or M or V,

X4=A or E or Q or R or S,

X5=Y or N or D,

X6=E or A or Q,

X7=A or D or E or H or K or S,

X8=F or A or D or E or H or Q or R or S,

X9=P or M,

X10=N or A or D or S or T or E or Q or R or I or V,

X11=S or A or D or E or H or K or N or P or Q or T,

X12=Y or H or D or G or H or N or R or S or T or wherein

X13=N or F or W or Y or H or K or R.

The above peptides according to formula 1 may be regarded as equivalentsor variants of the peptide according to SEQ ID NO: 34. In one embodimentof the invention, a variant of SEQ ID NO: 34 comprises 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2 or 1 amino acid substitution selected from the groupconsisting of substitutions X1, X2, X3, X4, X5, X6, X7, X8, X9, X10,X11, X12 and X13.

Accordingly, the region corresponding to SEQ ID NO: 16 may thereforealso be described as shown in formula 2:

Formula 2: (SEQ ID NO: 48) Y S E G X6 S X7 X8 X9 L X10 X11

wherein

X6=E or A or Q,

X7=A or D or E or H or K or S,

X8=F or A or D or E or H or Q or R or S,

X9=P or M,

X10=N or A or D or S or T or E or Q or R or I or V or wherein

X11=S or A or D or E or H or K or N or P or Q or T.

The above peptides according to formula 2 may be regarded as equivalentsor variants of the peptide according to SEQ ID NO: 16. In one embodimentof the invention, a variant of SEQ ID NO: 16 comprises 5, 4, 3, 2 or 1amino acid substitution selected from the group consisting ofsubstitutions X6, X7, X8, X9, X10 and X11.

The amino acids in bold print in formulas 1 and 2 represent the aminoacids in the peptides according to SEQ ID NO: 16 and SEQ ID NO: 34 (wildtype sequence) that should preferably not be changed or substituted. Thepreferred amino acids at positions X1-X13 are underlined. Theserepresent the wild type amino acids at the corresponding positions.

TABLE 1Human BMP-7 spanning consecutive 20-mer peptides with 2 amino acids overlapbetween peptides and their OA chondrocyte phenotype changing properties.Peptide sequence SEQ ID NO: ALP MMP13 ADAMTS5 COL10A COX-2 BAPX1/NKX3.2RUNX2 STGSKQRSQNRSKTPKNQEA 37 EALRMANVAENSSSDQRQAC 38 − − − − − − −ACKKHELYVSFRDLGWQDWI 39 − − − − − − − WIIAPEGYAAYYCEGECAFP 40 − − − − −− − FPLNSYMNATNHAIVQTLVH 41 − − − − − − − VHFINPETVPKPCCAPTQLN 42 − − −− − − − LNAISVLYFDDSSNVILKKY 43 − − − − − − − SSNVILKKYRNMVVRACGCH 44 −− − − − − − [−] denotes the absence of a significant effect or asignificantly decreased expression of chondrogenic marker BAPX1/NKX3.2or an increased expression of OA chondrocyte phenotypic markers ALP,MMP13, ADAMTS5, COX2, COL10A and RUNX2 in OA articular chondrocytes, incomparison to untreated OA articular chondrocytes.

TABLE 2Human BMP-7 derived consecutive 20-mer peptides with cysteine residues substituted toserine and their OA chondrocyte phenotype changing properties.Results are visualized in FIGS. 2-8. SEQ ID BAPX1/ Peptide sequence NO:ALP MMP13 ADAMTS5 COL10A COX-2 NKX3.2 RUNX2WIIAPEGYAAYYSEGESAFPLNSYMNATNHAIV  1 WIIAPEGYAAYYSEGESAFP  2 − − − − + −− IIAPEGYAAYYSEGESAFPL  3 − − − − + − − IAPEGYAAYYSEGESAFPLN  4 − − −− + − − APEGYAAYYSEGESAFPLNS  5 + − + + + + + PEGYAAYYSEGESAFPLNSY  6 +− + + + + + EGYAAYYSEGESAFPLNSYM  7 + + + + + + + GYAAYYSEGESAFPLNSYMN 8 + + + + + + + YAAYYSEGESAFPLNSYMNA  9 + + + + + + +AAYYSEGESAFPLNSYMNAT 10 + + + + + + + AYYSEGESAFPLNSYMNATN11 + + + + + + + YYSEGESAFPLNSYMNATNH 12 + + + + + + +YSEGESAFPLNSYMNATNHA 13 + + + + + + + SEGESAFPLNSYMNATNHAI 14 − − − − −− + EGESAFPLNSYMNATNHAIV 15 − − − − − − + YSEGESAFPLNS 16 + + + + + + +[+] denotes a significantly increased expression of chondrogenic markerBAPX1/NKX3.2 or a decreased expression of OA chondrocyte phenotypicmarkers ALP, MMP13, ADAMTS5, COX2, COL10A and RUNX2 in OA articularchondrocytes, in comparison to untreated OA articular chondrocytes.[−] denotes the absence of a significant effect or a significantlydecreased expression of chondrogenic marker BAPX1/NKX3.2 or an increasedexpression of OA chondrocyte phenotypic markers ALP, MMP13, ADAMTS5,COX2, COL10A and RUNX2 in OA articular chondrocytes, in comparison tountreated OA articular chondrocytes. A peptide was considered as havingOA chondrocyte phenotype suppressive activity when at least 6 out of 7markers were positive (+).

TABLE 3Human BMP-7 derived consecutive N-terminally and C-terminally truncatedpeptides with cysteine residues substituted to serine and their OAchondrocyte phenotype changing properties SEQ BAPX1/ Peptide sequenceID NO: ALP MMP13 ADAMTS5 COL10A COX-2 RUNX2 NKX3.2 RUNX2 YSEGESAFPLNS16 + + + + + + + APEGYAAYYSEGESAFPLNS 17 + − + + + + +PEGYAAYYSEGESAFPLNS 18 + − + + + + + EGYAAYYSEGESAFPLNS 19 + + + + + + +GYAAYYSEGESAFPLNS 20 + + + + + + + YAAYYSEGESAFPLNS 21 + + + + + + +AAYYSEGESAFPLNS 22 + + + + + + + AYYSEGESAFPLNS 23 + + + + + + +YYSEGESAFPLNS 24 + + + + + + + YSEGESAFPLNS 25 + + + + + + +YSEGESAFPLNSY 26 + + + + + + + YSEGESAFPLNSYM 27 + + + + + + +YSEGESAFPLNSYMN 28 + + + + + + + YSEGESAFPLNSYMNA 29 + + + + + + +YSEGESAFPLNSYMNAT 30 + + + + + + + YSEGESAFPLNSYMNATN 31 + + + + + + +YSEGESAFPLNSYMNATNH 32 + + + + + + + YSEGESAFPLNSYMNATNHA33 + + + + + + + 34 + + + + + + + 35 + + + + + + +EGYAAYYSEGESAFPLNSYMNATNHA 36 + + + + + + + [+] denotes a significantlyincreased expression of chondrogenic marker BAPX1/NKX3.2 or a decreasedexpression of OA chondrocyte phenotypic markers ALP, MMP13, ADAMTS5,COX2, COL10A and RUNX2 in OA articular chondrocytes, in comparison tountreated OA articular chondrocytes. [−] denotes the absence of asignificant effect or a significantly decreased expression ofchondrogenic marker BAPX1/NKX3.2 or an increased expression of OAchondrocyte phenotypic markers ALP, MMP13, ADAMTS5, COX2, COL10A andRUNX2 in OA articular chondrocytes, in comparison to untreated OAarticular chondrocytes. A peptide was considered as having OAchondrocyte phenotype suppressive activity when at least 6 out of 7markers were positive (+).

LEGEND TO THE FIGURES

FIG. 1: BMP-7 rescues OA-associated chondrocyte phenotype.

Osteoarthritic chondrocytes display a typical phenotype that ischaracterized by decreased expression of SOX9, COL2A1, ACAN andBAPX1/NKX3.2 and an increased expression of RUNX2, COL10A1, ALP, MMP13,ADAMTS5, COX-2 and IL-6 mRNAs (see left panel). BMP-7 (1 nM) is able torescue this OA-associated chondrocyte phenotype by normalizingexpression of above genes (left panel).

Functional ALP enzyme activity in cell lysates and PGE2 secretion in theculture medium was also normalized upon BMP-7 treatment of OAchondrocytes (right panel).

Expression of indicated mRNAs was determined by RT-qPCR, relatively tocontrol conditions (normalized for 28S rRNA expression). In graphs,error bars represent mean±SEM, statistical differences were calculatedas compared to healthy condition. *=p<0.05.

FIGS. 2-8: Actions of BMP-7 derived peptides on primary human OAarticular chondrocytes.

The action of 20-mer peptides (10 nM) of SEQ ID NO: 2-15 (peptides with1 amino acids intervals (19 amino acid overlap)) and SEQ ID NO:49 on theexpression of the indicated genes was determined on a validated pool ofpassage 2 OA human articular chondrocytes (n=18) and compared tofull-length recombinant BMP-7 (1 nM). Samples were harvested after 24hours and analyzed for indicated genes by RT-qPCR (corrected for 28SrRNA expression and relative to control conditions (no peptideexposure)). In the graphs, numbers on the x-axis represent individualpeptides according to their SEQ ID NO: error bars represent mean±SEM,statistical differences were calculated (one-way ANOVA with bonferronicorrection) to control condition. *=p<0.05 lower than control conditionfor COL10A1, ALP, RUNX2, COX-2, MMP13 and ADAMTS5 and higher thancontrol condition for BAPX1/NKX3.2. Random peptide is a peptideaccording to SEQ ID NO: 49 with amino acid sequenceSFILKKVLYDRVNDSANIYS.

FIG. 9: Diagram showing relative expression of COL2A1, Col10A1, COX-2and RUNX2 in isolated chondrocytes from OA patients in the presence of acontrol peptide, BMP-7 and a peptide according to SEQ ID NO: 16.

FIG. 10: Diagrams showing the relative expression of COL10A1 and MMP13in cartilage explants from OA patients in the presence of a controlpeptide, BMP-7 and a peptide according to SEQ ID NO: 16.

EXAMPLES Example 1: OA Chondrocyte Phenotype Suppressive Actions ofBMP-7 Peptides in the Presence of OA Synovial Fluid

We analysed candidate peptides from region A for their OA phenotypesuppressive actions on primary OA articular chondrocytes in the presenceof 20% (v/v) OA synovial fluid (SF). Data were combined from threeindividual OA chondrocyte isolates (n=3) that were each tested intriplicate. Seeded passage-2 OA chondrocytes were exposed to 100 nM ofpeptide in the absence or presence of OA synovial fluid and after 24hours analysed for mRNA expression of genes COL10A1, ALP, RUNX2, COL2A1,ACAN, SOX9, COX-2, IL-6, PGE, MMP13 and ADAMTS5 (corrected for 28S rRNAexpression and relative to control conditions (no peptide exposure)).

PGE2 secretion in culture supernatant was analysed by EIA and ALP enzymeactivity was determined by an in-house developed colorimetric assay forALP activity. GAG content was determined by colorimetric alcian blueassay.

Results were scored as + or − wherein + means that the peptide decreasedthe activity of genes COL10A1, ALP, RUNX2, COX-2, IL-6, PGE, MMP13 andADAMTS5 or increased the activity of COL2A1, ACAN and SOX9. For ALPactivity a + means a decreased activity and for GAG activity, a + meansan increased activity,

Example 2: Hypertrophy Suppressive Action of BMP-7 Peptides DuringChondrogenic Differentiation of ATDC5 Cells

ATDC5 cells were differentiated using standard protocols for 8 days inthe presence or absence of 1 nM peptides from region A. Peptides wereadded at start of differentiation and at every medium change (multiple)or only at start of differentiation and not during every medium change(single). At day 8 in differentiation, samples were harvested andanalysed for genes COL10A1, ALP, RUNX2, ACAN and BapX/Nkx3.2 by RT-qPCR(corrected for b-actin expression and relative to t=0).

Example 3: Evaluation of OA-Suppressive Actions in Ex Vixo Models for OA

Peptides for use according to the invention may also be investigated inex vivo testing: Full-thickness cartilage biopsies (3 mm punches) fromfemur condyles from TKA (K&L grade 2-3) may be freshly harvesteddirectly after surgery, randomized per patient and taken into ex vivoculture as described previously. Biopsies from patients may be exposedto selected peptides after twenty-four hours at 0.1, 1, 10 or 100 nM for7, 14 or 21 days and medium may be changed daily with fresh peptide.

BMP-7 at 1 nM may be used as positive control and vehicle or randompeptide may be used as negative control. After exposure, the cartilagebiopsies may be processed to address major cartilage quality-determiningparameters. DNA content per wet-weight and GAG content may bedetermined. ALP enzyme activity may also be investigated. PGE2 and GAGsecretion in the culture medium may be analyzed. Gene expression may bedetermined for major OA chondrocyte phenotype marker-genes as describedherein.

Local OA-related changes in the biopsies as a result of exposure to thepeptides may be analyzed by (immuno)histochemistry (alcian blue- andimmunostainings for ALP, COL10A1, RUNX2, COL2A1, SOX9 and COX-2) tofurther support the notion that the peptides as disclosed herein aresuitable for medical use in OA.

Diffusion and localization of the peptides into cartilage biopsies maybe addressed in a similar set-up wherein a biotinylated variant of thepeptides may be used. After culture these biopsies may be processed forhistochemistry. Peptide that is diffused into the cartilage biopsies maybe fluorescently visualized by Streptavidin-Alexafluor 488-mediateddetection. This approach may also aid in visualizing the phenotypiccontext and localization of the peptide by MALDI-IMS and help todiscriminate it from potential endogenous BMP-7 fragments.

Example 4: In Vivo Testing

Amino acid sequences of region-A are 100% homologous between human andmouse. In order to further establish the in vivo activity of thepeptides for use according to the invention, representative peptides maybe tested in a well-accepted model for post-traumatic OA, the DMM model.The medial meniscus may be destabilized in 12 weeks old C57BL/6 mice.One week after DMM induction, peptides may be administeredintra-articularly by twice-weekly injections as described previously.Dose may be based on intra-articular BMP-7 studies in which weeklyinjections of 250 ng BMP-7 in a rat knee joint (in 100 μl) showedfavorable outcomes. As 10 μl can be injected in an OA mouse joint anequivalent amount of 25 ng peptide in this volume may be injected perknee joint. An amount of 2.5 and 0.25 ng peptide may also be tested in 2additional groups to determine the pharmacological potency of thepeptide. Saline injections may be used as controls. The sample size ofthis experiment is advantageously 8 mice per group. Animals may besacrificed at consecutive time points after start of peptide treatment(2, 4, 8 weeks). Knee joints may be processed for (immuno)histochemicalanalyses and OARSI scoring (Safranin-O; modified Pritzker).

Example 5: Treatment of Isolated Chondrocytes from OA Patients

Isolated chondrocytes from OA patients (n=6) were treated with BMP-7 (1nM) or the 12-mer peptide according to SEQ ID NO: 16 (1 nM) for 24 h.Pro-chondrogenic (FIG. 9A) and hypertrophic (FIGS. 9B, 9C and 9D) geneexpression was determined via qRT-PCR and normalized for 28S rRNAlevels. These results confirmed our previous findings that BMP-7 or the12 mer induced an upregulation of pro-chondrogenic genes, such as Col2a1(A), and a downregulation of pro-hypertrophic genes, such as COL10A1,COX-2 and RUNX2 (B, C, D). These results show the BMP-7 mimickingbioactivity of the core sequence from the region-A peptide.

Example 6: Treatment of Cartilage Explants

4 mm2 cartilage explants were taken from non-lesion areas of OApatient's knee articular cartilage (n=5) and randomly assigned todifferent experimental treatment conditions (4 explants per treatmentgroup). After a 24 h equilibration period the explants were treated withBMP-7 (1 nM) or the 12-mer peptide according to SEQ ID NO: 16 (10 nM)for 24 h. Hypertrophic gene expression was determined via qRT-PCR andnormalized for 28S rRNA levels. After treatment with BMP-7 or the 12 merwe observed a downregulation of pro-hypertrophic genes, such as Col10a1(FIG. 10A) and MMP13 (FIG. 10B). These results are in line with theeffects described above and show the BMP-7 mimicking bioactivity of thepeptides according to the invention.

Example 7: Staining of Cartilage Explants

Cartilage explants obtained from 2 patients were cultured for 14 days inthe presence of BMP-7 (1 nM) or BMP-7 mimicking peptideGYAAYYSEGESAFPLNSYMN (SEQ ID NO: 8) at 10 nM. Glycosaminoglycans (GAGS),an important component of the extracellular matrix (ECM), were stainedwith Safranin-O (in red) and other tissues are counterstained with Fastgreen (in green/blue).

Both patients showed an increased Safranin-O intensity in BMP7 andpeptide GYAAYYSEGESAFPLNSYMN (SEQ ID NO: 8) treated explants compared tocontrol.

These results are in line with the effects described above and show theBMP-7 mimicking bioactivity of the peptides according to the invention.

Example 8: Treatment of Cartilage Explants

Cartilage explants of 3 mm² were taken from OA patient's knee cartilage(n=6) and treated with BMP-7 (1 nM), with peptide GYAAYYSEGESAFPLNSYMN(SEQ ID NO: 8) at 10 nM or a scrambled irrelevant control peptide (10nM) for 14 days. Prostaglandin E2 (PGE2) levels, a pro-hypertrophicfactor, were determined via an ELISA.

Synovial tissue samples from OA patients (n=6) were treated with BMP-7(1 nM), with peptide GYAAYYSEGESAFPLNSYMN (SEQ ID NO: 8) at 10 nM or ascrambled irrelevant control peptide (10 nM) for 24 h. PGE2 levels weredetermined via an ELISA.

Hoffa's fat pad tissue samples from OA patients (n=6) were treated withBMP-7 (1 nM) with peptide GYAAYYSEGESAFPLNSYMN (SEQ ID NO: 8) at 10 nMor a scrambled irrelevant control peptide (10 nM) for 24 h. PGE2 levelswere determined via an ELISA.

Meniscus tissue samples from OA patients (n=6) were treated with BMP-7(1 nM) with peptide GYAAYYSEGESAFPLNSYMN (SEQ ID NO: 8) at 10 nM or ascrambled irrelevant control peptide (10 nM) for 24 h. PGE2 levels weredetermined via an ELISA.

The results unequivocally showed reduce PGE2 levels in the cartilage andthe surrounding tissues after BMP-7 or peptide GYAAYYSEGESAFPLNSYMN (SEQID NO: 8) treatment. Treatment with the scrambled peptide did not resultin a reduction of PGE2 levels.

These results are in line with the effects described above and show theBMP-7 mimicking bioactivity of the peptides according to the invention.

REFERENCES

-   1. Glyn-Jones, S; Palmer, A J; Agricola, R; Price, A J; Vincent, T    L; Weinans, H; Carr, A J (3 Mar. 2015). “Osteoarthritis.”. Lancet    386: 376-87. doi:10.1016/S0140-6736(14)60802-3. PMID 25748615.-   2. Berenbaum F (2013). “Osteoarthritis as an inflammatory disease    (osteoarthritis is not osteoarthrosis!)”. Osteoarthritis and    Cartilage 21 (1): 16-21. doi:10.1016/j.joca.2012.11.012. PMID    23194896.-   3. March, L; Smith, E U; Hoy, DG; Cross, M J; Sanchez-Riera, L;    Blyth, F; Buchbinder, R; Vos, T; Woolf, A D (June 2014). “Burden of    disability due to musculoskeletal (MSK) disorders.”. Best practice &    research. Clinical rheumatology 28 (3): 353-66.    doi:10.1016/j.berh.2014.08.002. PMID 25481420.-   4. Maroudas A I (April 1976). “Balance between swelling pressure and    collagen tension in normal and degenerate cartilage”. Nature 260    (5554): 808-9. doi:10.1038/260808a0. PMID 1264261.-   5. Bollet A J, Nance J L (July 1966). “Biochemical Findings in    Normal and Osteoarthritic Articular Cartilage. II. Chondroitin    Sulfate Concentration and Chain Length, Water, and Ash Content”. J.    Clin. Invest. 45 (7): 1170-7. doi:10.1172/JCI105423. PMC 292789.    PMID 16695915.-   6. Brocklehurst R, Bayliss M T, Maroudas A, Coysh H L, Freeman M A,    Revell P A, Ali S Y (January 1984). “The composition of normal and    osteoarthritic articular cartilage from human knee joints. With    special reference to unicompartmental replacement and osteotomy of    the knee”. J Bone Joint Surg Am 66 (1): 95-106. PMID 6690447.-   7. Chou M C, Tsai P H, Huang G S, Lee H S, Lee C H, Lin M H, Lin C    Y, Chung H W (April 2009). “Correlation between the MR T2 value at    4.7 T and relative water content in articular cartilage in    experimental osteoarthritis induced by ACL transection”. Osteoarthr.    Cartil. 17 (4): 441-7. doi:10.1016/j.joca.2008.09.009. PMID    18990590.-   8. Grushko G, Schneiderman R, Maroudas A (1989). “Some biochemical    and biophysical parameters for the study of the pathogenesis of    osteoarthritis: a comparison between the processes of ageing and    degeneration in human hip cartilage”. Connect. Tissue Res. 19 (2-4):    149-76. doi:10.3109/03008208909043895. PMID 2805680.-   9. Mankin H J, Thrasher A Z (January 1975). “Water content and    binding in normal and osteoarthritic human cartilage”. J Bone Joint    Surg Am 57 (1): 76-80. PMID 1123375.-   10. Venn M, Maroudas A (April 1977). “Chemical composition and    swelling of normal and osteoarthrotic femoral head cartilage. I.    Chemical composition”. Ann. Rheum. Dis. 36 (2): 121-9.    doi:10.1136/ard.36.2.121. PMC 1006646. PMID 856064.-   11. Madry H, Luyten F P, Facchini A (2012). “Biological aspects of    early osteoarthritis”. Knee Surg. Sports Traumatol. Arthrosc. 20    (3): 407-22. doi:10.1007/s00167-011-1705-8. PMID 22009557.-   12. Englund M, Roemer F W, Hayashi D, Crema M D, Guermazi A (2012).    “Meniscus pathology, osteoarthritis and the treatment controversy”.    Nat. Rev. Rheumatol. 8 (7): 412-9. doi:10.1038/nrrheum.2012.69. PMID    22614907.-   13. Li G, Yin J, Gao J, Cheng T S, Pavlos N J, Zhang C, Zheng M H    (2013). “Subchondral bone in osteoarthritis: insight into risk    factors and microstructural changes”. Arthritis Research & Therapy    15 (6): 223. doi:10.1186/ar4405. PMID 24321104.-   14. Hill C L, Gale D G, Chaisson C E, Skinner K, Kazis L, Gale M E,    Felson D T (2001). “Knee effusions, popliteal cysts, and synovial    thickening: association with knee pain in osteoarthritis”. J.    Rheumatol. 28 (6): 1330-7. PMID 11409127.-   15. Felson D T, Chaisson C E, Hill C L, Totterman S M, Gale M E,    Skinner K M, Kazis L, Gale D R (3 Apr. 2001). “The association of    bone marrow lesions with pain in knee osteoarthritis”. Ann Intern    Med 134 (7): 541-9. doi:10.7326/0003-4819-134-7-200104030-00007.    PMID 11281736-   16. Sofat, N. Int J Exp Pathol 90, 463-479, (2009).-   17. Dreier, R. Arthritis Res Ther 12, 216, (2010).-   18. Tchetina, E. V. Arthritis 2011, 683970, (2011).-   19. van der Kraan, P. M. et. al. Osteoarthritis and cartilage 20,    223-232, (2012).-   20. Saito, A., Suzuki, Y., Ogata, S., Ohtsuki, C. & Tanihara, M.    Accelerated bone repair with the use of a synthetic BMP-2-derived    peptide and bone-marrow stromal cells. J Biomed Mater Res A 72,    77-82, doi:10.1002/jbm.a.30208 (2005).-   21. Chen, Y. & Webster, T. J. Increased osteoblast functions in the    presence of BMP-7 short peptides for nanostructured biomaterial    applications. J Biomed Mater Res A 91, 296-304,    doi:10.1002/jbm.a.32246 (2009).-   22. Caron, M. M. et al. Hypertrophic differentiation during    chondrogenic differentiation of progenitor cells is stimulated by    BMP-2 but suppressed by BMP-7. Osteoarthritis Cartilage 21, 604-613,    doi:10.1016/j.joca.2013.01.009 (2013).-   23. Caron, M. M. J. et al. BAPX1/NKX3.2 ACTS AS A CHONDROCYTE    HYPERTROPHY MOLECULAR SWITCH IN OSTEOARTHRITIS. Arthritis &    Rheumatology 67 (2015).-   24. Gentilucci, L., De Marco, R. & Cerisoli, L. Chemical    modifications designed to improve peptide stability: incorporation    of non-natural amino acids, pseudo-peptide bonds, and cyclization.    Curr Pharm Des 16, 3185-3203 (2010).-   25. Blaney Davidson, E. N. et al. Osteoarthritis Cartilage 23,    478-486, (2015).-   26. Glasson, S. S. et al. Nature 434, 644-648, (2005).-   27. Takayama, K. et al. Arthritis Res Ther 16, 482, (2014).-   28. Hayashi, et al. Arthritis Res Ther 10, R118, (2008).-   29. Sekiya, I. et al. J Orthop Res 27, 1088-1092, (2009).-   30. Pritzker, K. P. et al. Osteoarthritis Cartilage 14, 13-29,    (2006).-   31. Kirkwood, et al., J. Oral Implant. 24: 57-65 (2003)

The invention claimed is:
 1. A method of treating a subject at risk ofdeveloping osteoarthritis, the method comprising: administering to thesubject a peptide; wherein the peptide is between 12 and 28 amino acidsin length and comprises the full length of the amino acid sequenceaccording to SEQ ID NO: 16 or comprises the full length of a variant ofSEQ ID NO: 16 according to formula 2, wherein the amino acid sequence ofthe peptide is comprised in SEQ ID NO: 34 or a variant thereof accordingto formula 1, wherein formula 1 is: X1 X2 X3 G Y X4 A X5 Y S E G X6 S X7X8 X9 L X10 X11 X12 M N A T X13 H A (SEQ ID NO: 47) wherein X1=A or I orL or M or Y or V or E or H or K or Q or R, X2=P or Y or M, X3=E or R orH or K or N or P or Q or S or T or I or L or M or V, X4=A or E or Q or Ror S, X5=Y or N or D, X6=E or A or Q, X7=A or D or E or H or K or S,X8=F or A or D or E or H or Q or R or S, X9=P or M, X10=N or A or D or Sor T or E or Q or R or I or V, X11=S or A or D or E or H or K or N or Por Q or T, X12=Y or D or G or H or N or R or S or T, and X13=N or F or Wor Y or H or K or R; and wherein Formula 2 is: Y S E G X6 S X7 X8 X9 LX10 X11 (SEQ NO: 48) wherein X6=E or A or Q, X7=A or D or E or H or K orS, X8=F or A or D or E or H or Q or R or S, X9=P or M, X10=N or A or Dor S or T or E or Q or R or I or V, and X11=S or A or D or E or H or Kor N or P or Q or T.
 2. The method according to claim 1, wherein thepeptide comprises the amino acid sequence according to SEQ ID NO:
 16. 3.The method according to claim 1, wherein the amino acid sequence of thepeptide is fully comprised in SEQ ID NO:
 34. 4. A method of treating asubject at risk of developing osteoarthritis, the method comprising:administering to the subject a linear retro-inverso peptide orretro-inverso peptide; wherein the linear retro-inverso peptide orretro-inverso peptide is between 12 and 28 amino acids in length andcomprises the full length of the retro-inverso of the amino acidsequence according to SEQ ID NO: 16 or comprises the full length of theretro-inverso of a variant of SEQ ID NO: 16 according to formula 2,wherein the amino acid sequence of the linear retro-inverso peptide orretro-inverso is comprised in the retro-inverso of SEQ ID NO: 34 or avariant thereof according to formula 1, wherein formula 1 is:(SEQ ID NO: 47) X1 X2 X3 G Y X4 A X5 Y S E G X6 S X7 X8 X9 L X10X11 X12 M N A T X13 H A

wherein X1=A or I or L or M or Y or V or E or H or K or Q or R, X2=P orY or M, X3=E or R or H or K or N or P or Q or S or T or I or L or M orV, X4=A or E or Q or R or S, X5=Y or N or D, X6=E or A or Q, X7=A or Dor E or H or K or S, X8=F or A or D or E or H or Q or R or S, X9=P or M,X10=N or A or D or S or T or E or Q or R or I or V, X11=S or A or D or Eor H or K or N or P or Q or T, X12=Y or D or G or H or N or R or S or T,and X13=N or F or W or Y or H or K or R; and wherein Formula 2 is: Y S EG X6 S X7 X8 X9 L X10 X11 (SEQ ID NO: 48) wherein X6=E or A or Q, X7=Aor D or E or H or K or S, X8=F or A or D or E or H or Q or R or S, X9=Por M, X10=N or A or D or S or T or E or Q or R or I or V, and X11=S or Aor D or E or H or K or N or P or Q or T.
 5. The method according toclaim 1, wherein the subject suffers from at least one of synovialinflammation, synovial fibrosis, previous joint injury, abnormal jointor limb development, abnormal alignment of joints, one leg of adifferent length, high levels of joint stress, and cartilage loss.